Corynebacterium diphtheriae causes diphtheria. The bacterium produces a toxic protein, diphtheria toxin, which can be treated (e.g. using formalin or formaldehyde) to remove toxicity while retaining the ability to induce protective anti-toxin antibodies after injection. This treatment is referred to as “detoxification” or “toxoiding”, and the detoxified toxin is referred to as a “toxoid.” The diphtheria toxoids are used in diphtheria vaccines and are described in more detail in chapter 13 of the book “Vaccines” [1] and in chapter 31 of New Generation Vaccines [2].
Any therapeutic that is administered to humans and has been produced using a biological process has the potential for introducing harmful substances into the human body. Such harmful substances may be part of the medium used during the biological process. For example, animal-derived medium components such as fetal bovine serum bear the risk of containing aberrantly-folded proteins such as prions. Proteins derived from cow's milk have been suggested to cause severe allergic reactions in young children with cow's milk allergy, particularly after administration of DTP booster vaccines [3]. A similar risk may also exist in person with beef allergy [4]. Traditionally, diphtheria toxoid was obtained by growing C. diphtheriae in growth medium containing animal-derived components (e.g. in Linggoud & Fenton medium), such as bovine extract and/or casamino acids derived from cow milk.
The use of proteinaceous material of non-animal origin removes this risk. EP-B-1849860 discloses a medium for cultivating C. diphtheriae comprising at least 20% by dry weight of a non-animal proteinaceous material which is a yeast extract [5]. WO2005/056773 discloses a C. diphtheriae culture medium for the production of diphtheria toxin, which is substantially free of animal-derived components, and methods for producing the toxin [6]. WO2006/042542 discloses a fermentation medium for producing bacterial toxins using a non-animal and non-soya derived protein source [7]. WO00/50449 discloses a method of purifying diphtheria toxin comprising fermenting a microorganism strain capable of producing diphtheria toxin using glucose as a carbon source. In a preferred embodiment, this patent application discloses the use of a growth medium containing no more than 1% yeast extract [8].
Although these media are based on non-animal derived protein sources, and so can decrease contamination risks, none of them results in high yields of diphtheria toxin during industrial production (e.g. using fermenters in the 100-600 L range), and low yields are a drawback of these processes. Accordingly, none of these processes yield a cross-linked diphtheria toxoid free from animal-derived components with high enough potency to render the diphtheria toxoid suitable for human vaccine production.
It is thus an object of the invention to provide further and improved fermentation media suitable for use in industrial-scale, high-yield manufacturing of diphtheria toxin for production of a high potency vaccine suitable human administration.
In traditional processes to prepare diphtheria toxoid, the toxin is treated with formaldehyde in the presence of culture medium components e.g. see FIG. 4 of chapter 31 in reference 2. As well as cross-linking and detoxifying the diphtheria toxin, the formaldehyde causes covalent cross-linking of the medium components. This cross-linking means that the animal-derived components, if present in a growth medium, can be irreversibly locked into a human vaccine product. Higher purity toxoids for vaccine use can be obtained by purifying the toxin before the formaldehyde treatment. Patent Application GB-969772 discloses a method for producing toxoids from diphtheria toxin, comprising treating the toxin in an aqueous medium with formaldehyde in the presence of an aliphatic diamine of molecular weight below 200 which contains a primary or secondary amino group [9]. Frech et al. [10] discloses a physiochemical analysis of two purified diphtheria toxoids: the first was prepared by a conventional process in which the diphtheria toxin was formalinised and then purified; the second was first highly purified and then detoxified. WO2005/056773 discloses detoxification of an at least 75% pure diphtheria toxin. Even if high purity is achieved in a purification step preceding detoxification, however, residual animal-derived components of the fermentation medium used to prepare the diphtheria toxin (e.g. proteins, polypeptides, peptides and amino acids) are cross-linked by formaldehyde to the diphtheria toxoid obtained during the detoxification step.
It is a further object of the invention to provide a diphtheria toxoid that is free from crosslinked animal-derived components. It is another object of the invention to provide further improved processes in which diphtheria toxin is first highly purified and then detoxified.
Metz et al. [11] demonstrate that glycine and formaldehyde concentrations during detoxification of diphtheria toxin affects the antigenic properties of the resulting diphtheria toxoids. In particular, the formaldehyde concentration used during detoxification directly influences the immunogenicity of diphtheria toxoids leading to up to a 15-fold difference in potency between different diphtheria toxoid preparations.
An additional object of the invention is to provide a diphtheria toxoid that is free from crosslinked animal-derived components that has a consistently high potency and can be employed in the preparation of vaccines suitable for use in humans.